Methods of identifying respondents to hypoxia inducible factor 1-alpha inhibitors

ABSTRACT

This invention relates to methods of measuring HIF expression and activity, as well as measuring inhibition of HIF following administration of an HIF inhibitor useful in treating HIF related diseases. The present invention further relates to methods of identifying individuals who will respond to HIF inhibitors. The invention also relates to methods of monitoring a patient response to a given dosage of an HIF inhibitor. The invention also includes assays and kits for performing the methods described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/379,034,filed Apr. 17, 2006, which is a continuation in part of U.S. applicationSer. No. 10/929,156, filed Aug. 30, 2004, now U.S. Pat. No. 7,399,785,and claims the benefit of U.S. Provisional Application No. 60/671,765,filed Apr. 15, 2005; the contents of all of the aforementionedapplications are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

NOT APPLICABLE

JOINT RESEARCH AGREEMENT

NOT APPLICABLE

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A CD

NOT APPLICABLE

BACKGROUND OF THE INVENTION

Hypoxia inducible factor-1 (HIF-1) plays a central role in thedevelopment and progression of tumors. While not wishing to be bound bytheory, it appears this is because HIF-1 controls the expression of morethan 40 target genes whose protein products play crucial roles inallowing the survival of cells under adverse environmental conditionsand in response to radiation or chemotherapy. These include the geneencoding VEGF, which is required for tumor angiogenesis, insulin-likegrowth factor 2 (IGF2), which promotes tumor cell survival, and glucosetransporters 1 and 3, and glycolytic enzymes such as aldolase A and C,hexokinase 1 and 3, lactate dehydrogenase A and PGK.

HIF-1α is a subunit of HIF-1. HIF-1α protein is found in a wide varietyof human primary tumors but only at very low levels in normal tissue.The importance of HIF-1α to cancer is demonstrated by the high incidenceof tumors such as renal cell carcinoma, pheochromocytoma andhemingioblastoma of the central nervous system in individuals with lossof function of both alleles of the VHL gene leading to elevated HIF-1αlevels. In addition, most cases of sporadic renal cell carcinoma areassociated with an early loss of function of the VHL gene and increasedHIF-1α levels. Reintroduction of the intact VHL gene into cells derivedfrom renal carcinomas restores HIF-1α to normoxic levels and decreasestumorigenicity. HIF-1α levels are also increased in cancer cells withmutant or deleted PTEN.

Many human tumors have been shown to overexpress HIF-1α protein as aresult of intratumoral hypoxia and genetic alterations affecting keyoncogenes and tumor suppressor genes. In addition, over-expression ofHIF-1α correlates with treatment failure and mortality. However, loss ofHIF-1 activity has dramatic negative effects on tumor growth,vascularization and energy metabolism in xenograft assays. Thereforeinhibition of HIF-1 represents a promising new approach to cancertherapy since its inhibition may lead to the selective killing of tumorcells over normal cells.

The HIF-1α inhibitor PX-478(S-2-amino-3-[4′-N,N,-bis(2-chloroethyl)amino]phenyl propionic acidN-oxide dihydrochloride) inhibits growth of hypoxic tumor cells invitro. PX-478 inhibits HIF-1α protein, leading to decreased HIF-1transactivation and expression of the downstream target gene VEGF.PX-478 also decreases HIF-1α in vivo at a non-toxic dose. Interestingly,this inhibition has been shown to occur independently of the VHLpathway, the most well-studied mechanism for controlling HIF-1αstabilization.

Many other factors have been shown to affect HIF-1α protein, includingthe P53 tumor suppressor pathway as well as oncogenes signalling throughthe PI3K and MAPK pathways. Several recent studies have also reportedindirect inhibition of the HIF-1 pathway in a VHL independent manner.These include inhibition of PI3K using LY294002, inhibition of themolecular chaperone HSP90 using geldanaycin, and inhibition of redoxsignalling by PX-12 (1-methylpropyl 2-imidazolyl disulfide) andpleurotin. Indeed, thioredoxin reductase activity was shown in thisstudy to be significantly decreased at concentrations of PX-478 whichcorrelate well with HIF-1α inhibition.

A recent study has shown that Trx-1 binds to, and inhibits, thetumor-suppressor protein PTEN leading to activation of the PI3K pathwaythrough AKT. In light of the findings that the PI3K/AKT pathway isinvolved in the stabilization and activation of HIF and that the PI3Kinhibitor LY294002 also decreases HIF-1α protein in a VHL independentmanner, it is possible Trx-1 may affect HIF-1α through this pathway.Recent studies suggest that this is cell-type dependent and, whenobserved, lies downstream of HIF activation or in a parallel pathway.

One goal of targeted therapies for disease treatment, such as the HIF-1αinhibitor PX-478 in treating cancer, is to be able to select patientsthat are most likely to respond to the drug. In the case of cancer,while immunohistochemical techniques verifying the upregulation ofHIF-1α in the target tumor is the gold standard, often invasiveprocedures such as tumor biopsies are not possible and the tumor tissuerequired for such tests are not available. Non-invasive techniques havebeen explored to evaluate the effect of PX-478 on HIF-1α levels in theclinical setting, including the use of Dynamic ContractEnhanced-magnetic resonance imaging (DCE-MRI) and diffusion weighted(DW) MRI to evaluate tumor vascular permeability. It is possible thateven though a tumor may express some level of HIF-1α, the protein somepatients express may not be responsive to a synthetic inhibitor such asPX-478. In order to provide effective treatment to an individual, itwould be helpful to identify those individuals who will be responsive orsusceptible to an HIF inhibitor (e.g., PX-12, PX-478, 2-ME2). It wouldbe useful to be able to identify patients with hypoxic tumors andcorresponding increased HIF-1α levels, and to be able to evaluate theHIF-1α inhibition following therapy.

SUMMARY OF THE INVENTION

In one embodiment, a method of identifying an individual susceptible totreatment with a HIF-1α inhibitor is provided. The method may comprisethe steps of obtaining a blood sample from said individual; exposing theblood sample to a leukocyte stimulating agent to the blood sample;measuring a second level of HIF in the blood sample; and identifying anindividual who is susceptible to treatment with a HIF-1α inhibitor,wherein elevated levels of HIF indicate said individual's susceptibilityto said treatment with said HIF-1α inhibitor. In preferred embodiments,the HIF-1α inhibitor is PX-478 or PX-12. In another embodiment, themethod may further comprise the step of separating or isolating PBMCfrom the blood sample. In another embodiment, the method may furthercomprise the step of inducing a hypoxic condition in the blood sample.

In a further embodiment, a method of determining efficacy of a HIF-1αinhibitor therapy is provided. The method may comprise the steps ofobtaining a first blood sample from said individual; exposing the firstblood sample to a first leukocyte stimulating agent; measuring a levelof HIF in said first blood sample; and identifying said individualsusceptible to treatment, wherein elevated levels of HIF indicate saidindividual's susceptibility to said treatment with said HIF-1αinhibitor; administering a HIF-1α inhibitor to said individual;obtaining a second blood sample from said individual; exposing saidsecond blood sample to a second leukocyte stimulating agent; measuring asecond level of HIF expression in said blood sample, wherein decreasedlevels of HIF indicates effective HIF-1α inhibitor therapy.

In another embodiment, a method of determining efficacy of a HIF-1αinhibitor therapy in an individual is also provided. The method maycomprise obtaining a first blood sample from said individual; measuringa first level of HIF in said blood sample; administering a HIF-1αinhibitor to said individual; obtaining a second blood sample from saidindividual; exposing said blood sample to a leukocyte stimulating agentand hypoxic conditions; and measuring a second level of HIF in saidsecond blood sample, wherein decreased levels of HIF indicates effectiveHIF-1α inhibitor therapy.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of necessary fee.

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of theinvention, which follows:

FIGS. 1A and 1B illustrate the effect of PX-478 on HIF-1α and HIF-1αprotein levels;

FIG. 2 illustrates the recovery of HIF-1α protein after inhibition byPX-478;

FIGS. 3A, 3B, and 3C illustrate the effect of PX-478 on HIF-1 in vivo;

FIG. 4 illustrates DFO and CoCl₂ can induce HIF-1α upregulation innormal leukocytes (PBMC) and leukemic T cells (Jurkat);

FIGS. 5A and 5B illustrate PX-478 exposure decreases HIF-1α expressionin stimulated/hypoxic PBMC and Jurkat cells and can inhibit hypoxiainduced accumulation of the HIF-1α protein;

FIGS. 6A and 6B illustrate HIF-1α upregulation and inhibition by PX478can be quantified by flow cytometry;

FIGS. 7A and 7B illustrate HIF-1α but not HIF-1β is down regulated byPX478 in freshly isolated PBMC as measured by flow cytometry;

FIGS. 8A and 8B illustrate a rapid and dose dependent decrease in HIF-1αdue to PX-478 spiked into human whole blood;

FIG. 9 illustrates stimulation of whole blood with PMA and ionophorealone in responders and a non-responder, as measured by flow cytometry;

FIG. 10 illustrates stimulation of PBMCs with PMA and ionophore alone ina non-responder, as measured by Western analysis;

FIG. 11 illustrates treatment of stimulated responder and non-responderPBMCs with PX-478;

FIGS. 12A and 12B illustrate HIF-1α expression in stimulated andunstimulated cancer patient total lymphocyte population and in the Tcell and B cell subsets.

FIGS. 13A and 13B illustrate HIF-1α expression in normal donors'stimulated PBMCs treated with two different HIF-1α inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

One aspect of the present invention relates to assays to measureexpression or activity levels of an HIF, preferably HIF-1α. In certainembodiments, the assay comprises obtaining a sample of an individual'speripheral blood, inducing a hypoxic condition in at least one cell ofthe sample, and measuring the response of one or more of theindividual's peripheral blood mononuclear cells (PBMCs) or leukocytes tothe hypoxic condition. In a preferred embodiment, the assay comprisesmeasuring the response in a population or subset of PBMC or leukocytesobtained from an individual's blood to a hypoxic condition. In certainembodiments, the assay comprises obtaining a sample of an individual'speripheral blood, separating one or more cells obtained from apopulation of the individual's leukocytes from the blood sample,inducing a hypoxic condition in the cell or cells, and measuring theresponse of the cell or cells to the hypoxic condition. In furtherembodiments, the assay comprises measuring the response of the cell orcells to HIF inhibitors, including, for example, N-oxide and derivativesthereof, and agents which inhibit HIF through inhibition of otherpathways including, but not limited to, inhibition of thioredoxin orthioredoxin reductase, PI3 kinase or AKT.

The cell or cells obtained from a population of PBMCs or leukocytes mayinclude one or more types of blood cells, for example, one or more of Tcells, B cells, NK cells, and monocytes, in any combination. In oneembodiment, the method of the invention comprises measuring a responseto stimulation and hypoxia in at least one leukocyte. In anotherembodiment, the method of the invention comprises measuring a responseto stimulation and hypoxia in at least one T cell.

According to the invention, the method comprises measuring a response inone or more blood cells of an individual to a hypoxic condition. Themeasuring step generally involves detecting the expression or activityof an HIF. The measuring step may include, for example, measuringexpression or activity of an HIF-1 protein, or may include measuringexpression of an HIF gene in one or more blood cells. The measuring stepmay be performed by a variety of methods, including, for example, byflow cytometry.

A “hypoxic condition” refers to a state of oxygen deficiency sufficientto cause impairment of function. Preferably a hypoxic condition iscreated by exposure to about 1% oxygen. Cells are normally exposed toabout 20% oxygen. A hypoxic condition can be induced in blood cells by avariety of methods known in the art, including methods demonstrated inthe Examples, below, including but not limited to administration orexposure to DFO.

In further aspects, the invention relates to identifying subjects thatmay be responsive to HIF inhibitors, for example PX-478, and otherN-oxides or derivatives thereof, PX-12, 2-ME2, topotecan andcamptothecin or derivatives thereof. HIF inhibitors include agents thatinhibit HIF, e.g., agents that inhibit HIF-1α. Such inhibition may bedirect or indirect. That is, the inhibitor may act directly on an HIFgene or protein molecule to inhibit its expression or activity. However,the inhibitor may act indirectly by affecting expression or activity ofanother molecule which ultimately leads to inhibition of expression oractivity of an HIF gene or protein molecule. In certain aspects, therapyincludes administration of an HIF inhibitor, including, but not limitedto, an HIF inhibitor discussed herein or any of the HIF inhibitorsdiscussed in Powis, G. and Kirkpatrick, L., Hypoxic inducible factor-1αas a cancer drug target, Mol. Cancer Ther. 3(5):647-654 (2004), which ishereby incorporated by reference in its entirety. The invention,however, is not limited to evaluating HIF inhibition caused by known HIFinhibitors; rather, it can be applied to evaluate any HIF inhibitor.

Diseases that may be treated by directly or indirectly inhibiting HIF,particularly HIF-1α, include diseases associated with angiogenesis orneovascularization. Diseases associated with HIF, or cells relying onHIF for survival, which may be treated with HIF inhibitors includecancer, ischemic myocardial and limb disease, ischemic stroke,Alzheimer's disease, choroidal and retinal neovascularization,age-related macular degeneration, joint disease, inflammation,neurodegenerative diseases, autoimmunity, infectious disease andischemic reperfusion injury.

According to some aspects of the invention, the method further comprisestreating at least one leukocyte of a blood sample from an individualwith an HIF inhibitor prior to measuring the response of the at leastone leukocyte to a hypoxic condition. The HIF inhibitor may be one of avariety of HIF inhibitors as discussed herein. In one embodiment, theHIF inhibitor is PX-478. In other embodiments, the HIF inhibitor isPX-12.

The invention comprises, in some aspects, obtaining a first subset of atleast one leukocyte from a blood sample, obtaining a second subset of atleast one leukocyte from the blood sample, and treating either the firstor second subset with an HIF inhibitor. According to this embodiment,the measuring step then comprises measuring HIF protein level oractivity in the two subsets. The invention may further comprisecomparing the HIF protein level or activity of the first and secondsubsets.

Generally, a normal donor's PBMCs, once stimulated with phorbolmyristate acetate (PMA) and calcium ionophore A23187, will upregulateHIF-1α expression under normoxic conditions. This HIF-1α accumulationcan typically be enhanced under hypoxia and is sensitive to PX-478exposure. However, as described in the Examples, we have identifieddonors whose PBMCs did not upregulate HIF-1α upon stimulation alone andrequired a second signal such as hypoxia to visualize HIF-1αaccumulation by flow cytometry. The addition of PX-478 added directly tothe donor's whole blood one hour before the isolation of the PBMC andsubsequent stimulation with PMA/ionophore and DFO had no effect on theHIF-1α levels under conditions that lowered the HIF-1α expression of twonormal donors by at least 50%.

Therefore, we have developed assays capable of distinguishing betweenindividuals whose HIF-1α levels in PBMCs can be stimulated and thatrespond to PX-478 or other HIF inhibitors, versus those whose cells arenot responsive. The HIF-1α response of the PBMCs to stimulation andhypoxia, and/or the response to PX-478 using such an assay provides atest a priori that can identify patients that are likely to benefit bytreatment with an HIF-1α inhibitor. Additionally, we have developed anassay to monitor patient response to an HIF inhibitor.

A further aspect of the present invention is to clinically evaluate HIFinhibition, e.g., HIF-1α inhibition, following therapy. One embodimentcomprises, therefore, a method of monitoring patient response to a HIFinhibitor. The method comprises obtaining a first sample of a patient'sblood, treating the patient with an HIF inhibitor, obtaining a secondsample of the patient's blood, inducing a hypoxic condition in the bloodsamples, measuring a response to the hypoxic condition in at least oneleukocyte of the blood samples, and comparing the responses of the bloodsamples. Depending on the response observed, a patient's therapy using agiven HIF inhibitor may be modified, for example, the dosage may beincreased or decreased or the frequency of dosing may be increased ordecreased.

In a further embodiment, a method of identifying an individualsusceptible to treatment with a HIF-1α inhibitor is provided. The methodmay comprise the steps of obtaining a blood sample from said individual;measuring a first level of HIF in the blood sample; administering a Tcell stimulating agent to the blood sample; measuring a second level ofHIF in the blood sample; and identifying an individual who issusceptible to treatment with a HIF-1α inhibitor, wherein elevatedlevels of HIF indicate said individual's susceptibility to saidtreatment with said HIF-1α inhibitor. In preferred embodiments, theHIF-1α inhibitor is PX-478 or PX-12. In another embodiment, the methodmay further comprise the step of inducing a hypoxic condition in theblood sample.

The leukocyte stimulating agent may be any agent capable of stimulatingT cells and/or B cells. For example, the leukocyte stimulating agent maybe an antibody or a mitogen. Mitogens are agents that are able to inducecell division (mitosis) in T cells and/or B cells. Preferably mitogensinclude lectins, such as concanavalin A (Con A), phytohemagglutinin(PHA) and pokeweed mitogen (PWM), and lipopolysaccharide or endotoxin(LPS). Superantigens are potent T cell mitogens, which may also be a Tcell stimulating agent. Exemplary superantigens include staphylococcalexotoxins, such as staphylococcal toxic shock syndrome toxin 1 andstaphylococcal enterotoxins A and B (SEA and SEB, respectively. Inpreferred embodiments, the leukocyte stimulating agent may be selectedfrom phorbol myristate acetate, calcium ionophore, concanavalin A,anti-CD3 antibody, anti-CD28 antibody, PHA and combinations thereof.

HIF levels may be measured by measuring levels of HIF protein expressionor HIF gene expression. Preferably, the step of measuring comprisesmeasuring expression of a HIF-1α protein. Elevated levels of HIF areabout at least a 2-fold increase of HIF over a baseline level. Baselinelevels of HIF may be obtained from the specific individual or from anaverage of normal levels of HIF.

In another embodiment, a method of determining efficacy of a HIF-1αinhibitor therapy is provided. The method may comprise the steps ofobtaining a first blood sample from said individual; measuring a firstlevel of HIF in said blood sample; exposing said blood sample to a firstleukocyte stimulating agent; measuring a second level of HIF in saidfirst blood sample; and identifying said individual susceptible totreatment, wherein elevated levels of HIF indicate said individual'ssusceptibility to said treatment with said HIF-1α inhibitor;administering a HIF-1α inhibitor to said individual; obtaining a secondblood sample from said individual; administering a second T cellstimulating agent; measuring a third level of HIF expression in saidblood sample, wherein decreased levels of HIF indicates effective HIF-1αinhibitor therapy.

A method of determining efficacy of a HIF-1α inhibitor therapy in anindividual is also provided. The method may comprise obtaining a firstblood sample from said individual; measuring a first level of HIF insaid blood sample; administering a HIF-1α inhibitor to said individual;obtaining a second blood sample from said individual; exposing saidblood sample to a leukocyte stimulating agent and hypoxic conditions tosaid blood sample; and measuring a second level of HIF in said secondblood sample, wherein decreased levels of HIF indicates effective HIF-1αinhibitor therapy.

The invention further comprises kits or assays. In one embodiment, a kitor assay comprises a component for obtaining a blood sample from anindividual, a component for inducing a hypoxic condition in the bloodsample or subset thereof, a component for measuring a response to saidhypoxic condition in the blood sample or subset thereof, and a componentfor identifying an individual susceptible to treatment.

According to another embodiment, the kit or assay of the inventioncomprises a component for obtaining a blood sample from an individual, acomponent for separating one or more leukocyte cells from the blood, acomponent for inducing a hypoxic condition in said one or more leukocytecells, a component for measuring a response to hypoxia in the one ormore leukocyte cells, and a component for identifying said individualsusceptible to treatment. The kit or assay of the invention may alsoinclude a component for measuring expression of an HIF-1α protein insaid at least one leukocyte. The kit or assay of the invention may alsoinclude a component for measuring expression of HIF-1α protein usingflow cytometry.

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention. Various modifications and combinations of the illustrativeembodiments, as well as other embodiments of the invention, will beapparent to persons skilled in the art upon reference to thedescription.

Example 1

Cell culture and hypoxia treatment. MCF-7 human breast cancer and HT-29colon cancer cells were obtained from the American Tissue TypeCollection. Cells were grown under humidified 95% air, 5% CO₂ incubatorat 37° C. in Dulbecco's modified Eagle's medium (DMEM) supplemented with10% fetal bovine serum (FBS), and 1 mg/ml G418 where appropriate. Forexposure to hypoxia the culture flasks were incubated for various timesin a humidified chamber at 37° C. with a gas mixture containing 5%CO₂/74% N₂/21% argon. Oxygen levels were kept at 1% in the gas phaseusing an oxygen sensor (Pro:Ox 110, Biospherix, Redfield, N.Y.). At theend of the study cells were washed twice with ice cold phosphatebuffered saline, pH 7.5 (PBS). One ml of media from each flask wasremoved after treatment and stored at −80° C. for measurement of VEGFlevels.

Cell growth and viability assays. Growth inhibition assays were carriedout using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. For analysis under hypoxic conditions, plates wereincubated for 16 h in 1% oxygen in the presence of the drug then placedin 20% oxygen for the remainder of the 72 h.

Immunoblotting. Nuclear and cytoplasmic extracts were prepared usingNE-PER™ Nuclear and Cytoplasmic Extraction Reagents according to themanufacturer's instructions. Western blotting was performed using mouseanti-human HIF-1α (1 μg/ml); mouse anti-human HIF-1β (1 μg/ml); mouseanti-human iNOS (5 μg/ml); and goat anti-human lamin A (0.5 μg/ml).Anti-mouse or anti-goat horseradish peroxidase-conjugated secondaryantibodies were used at a dilution of 1:5000 for detection bychemiluminescence and blots were quantified using ImageQuant software.

Immunohistochemistry. Human breast carcinoma MCF-7 cells (10⁷ cells inmatrigel per mouse) were injected subcutaneously into the flanks of SCIDmice implanted with estrogen pellets. Tumors were allowed to grow to 0.5g. The mice then received intraperitoneal vehicle alone or 120 mg/kgPX-478. Four hours later the tumors were excised, fixed in formalin andembedded in paraffin. Sections were stained with antibodies to HIF-1α(10 μg/ml; Transduction Labs) or VEGF (7 μg/ml) using an automatedimmunostainer system. Staining was quantified using Simple PCI analysissoftware.

PX-478 shows inhibition in hypoxia and normoxia. Human breast carcinomaand human colon carcinoma HT-29 cells were treated for 16 hr withvarying concentrations of PX-478 in the presence of normoxia (20%oxygen) or hypoxia (1% oxygen). The cells were then washed three timeswith warm drug-free medium and incubated for the remainder of 72 hr. TheMTT assay was then carried out to determine growth inhibition. Data inTable 1 represent the mean±SE from three experiments carried out induplicate. PX-478 shows growth inhibition under hypoxia (1% oxygen) andnormoxia (20% oxygen) (p=<0.01) with a ratio of growth inhibition underhypoxia to that in normoxia at 1.25 in MCF-7 cells and 1.20 in HT-29cells.

TABLE 1 IC₅₀ (μM) CELL LINE Normoxia Hypoxia MCF-7 25.1 ± 1.5 20.0 ± 2.0HT-29 29.5 ± 2.4 23.9 ± 2.3

PX-478 inhibits HIF-1α protein. HIF-1α is a key controller of thecellular response to hypoxia. Therefore, we examined the effect ofPX-478 on HIF-1α protein levels. As shown in FIG. 1, PX-478 inhibitshypoxia-induced (1% oxygen) HIF-1α protein in human breast carcinomaMCF-7 (FIG. 1A) and human colon carcinoma HT-29 cells (FIG. 1B), withIC₅₀ values of 3.5±2.0 and 17.8±5 μM respectively. HIF-1α protein levelswere very low under normoxia (20% oxygen) as reported previously so noeffect was seen. No effect of PX-478 was seen on HIF-1β levels (FIGS. 1Aand 1B).

Example 2

HIF-1α protein remains inhibited for up to 4 h after removal of PX-478.To investigate how long HIF-1α protein remains inhibited after treatmentof cells with PX-478, MCF-7 cells, as shown in FIG. 2, were treated withPX-478 25 μM for 16 h, the drug was then washed out and recovery ofHIF-1α was measured. Nuclear cell extracts were prepared at the timepoints indicated and Western blotting was performed to measure levels ofHIF-1α protein. Levels of HIF-1α protein after 16 h under normoxia (20%oxygen; N) are also shown as a control. HIF-1α protein levels returnedto pre-treatment levels within 4 h of removal of the drug.

Example 3

PX-478 inhibits HIF-1α protein in vivo. MCF-7 cells were grown asxenografts in the flanks of SCID mice. When the xenografts reached 0.3 gthe mice were treated with vehicle control or 100 mg/kg PX-478 (FIGS. 3Aand 3B respectively). Four hours later tumors were removed, formalinfixed, embedded in paraffin and the level of HIF-1α protein was measuredusing immunohistochemistry (FIGS. 3A and 3B). In FIG. 3C, the intensityof HIF-1α staining was quantified using Simple PCI software. Theasterisk (*) shows a significant difference from controls (p=<0.01).Data represent the mean±SE. As shown in FIGS. 3A and 3B, treatment ofMCF-7 cells grown as xenografts in the flanks of SCID mice showedsignificantly decreased levels of HIF-1α protein after 4 hr treatmentwith PX-478 (p=<0.005). Staining remained nuclear in localization evenin PX-478 treated cells but levels of HIF-1α were decreased to 50% inPX-478 treated mice compared to untreated controls as shown in FIG. 3C.

Recently, several drugs have been reported to indirectly inhibit theHIF-1 complex. As mentioned above, the HSP90 inhibitor geldanamycin hasbeen reported to inhibit HIF-1α protein by a pVHL-independent mechanism.A number of camptothecin analogues have also been identified asinhibitors of HIF-1α protein and transactivation using a high-throughputscreening approach. It is not clear if these compounds simply inhibitgeneral transcription via topoisomerase I inhibition or act as specificHIF-1 inhibitors. DX-2-1 (a carbomycin derivitive) was also identifiedusing the same screen but is known to affect a number of transcriptionfactors in addition to HIF-1. We have previously identified twoinhibitors of the thioredoxin-1 redox system, PX-12 and pleurotin, asinhibitors of HIF-1α protein, HIF-1 transactivation and hypoxia-inducedVEGF production in vitro and HIF-1α protein in vivo. Treatment methodsusing any HIF inhibitor, including any of the drugs discussed herein,would benefit from the present invention to help determine aphysiologically effective dose and to select patients that will mostbenefit from treatment.

The effect of PX-478 against Panc-1 human pancreatic cancer as MCF-7Human Breast Cancer; Human Prostate Cancer and HT-29 Colon Cancer werepreviously shown in U.S. Ser. No. 10/929,156, filed Aug. 30, 2004, theentirety of which is hereby incorporated by reference. The effect ofPX-478 on HT-29 Tumor xenograph a HIF-1α and on plasma VEGF levels werealso shown in U.S. Ser. No. 10/929,156.

Example 4 Jurkat and PBMC Activation and DFO Treatment Protocol forHIF-1α Induction

Hypoxia induction: PBMC. Peripheral Blood Mononuclear Cells (PBMCs) wereisolated from heparinized blood of healthy volunteers usingHistopaque-1077 (Sigma-Aldrich) density gradient centrifugation. Toinduce leukocyte activation, PBMC were incubated overnight in AIMV mediasupplemented with 5% FCS, 5 ng/mL IL-15, 100 U/mL IL-2 (Peprotech, RockyHill, N.J.) in the presence or absence of 1 or 50 ng/mL phorbolmyristate acetate (PMA) (EMD Biosciences, La Jolla, Calif.) and 250ng/mL calcium ionophore A23187 (Sigma). To simulate hypoxia, 50 μM DFOwas added for 16 hr. The PBMCs were then harvested and analyzed by flowcytometry or Western for HIP-1α protein expression.

Western Blot: Standard Western blot procedures were used to analyzenuclear extracts (10 μg/lane) on a 12% gel, transferred to PVDF membraneand immunoblotted with 1 μg/mL rabbit polyclonal anti-HIF-1α antibodyH-206 (Santa Cruz Biotechnology, Santa Cruz, Calif.) and developed withSuperSignal® West Dura Extended Duration Substrate kit (Pierce). Theimmunocomplexes were visualized on a Kodak Image Station 440 CF (EastmanKodak Company, Rochester, N.Y.).

Results. Upregulation of HIF-1α in primary stimulated PBMC or the acuteleukemia tumor cell line Jurkat, can be induced in vitro usingdeferoxamine (DFO), an iron chelator or Cobalt chloride (CoCl₂). PBMCwere left unstimulated or were stimulated with PMA (50 ng/mL) and thecalcium ionophore A23187 (250 ng/mL) for 16 hours in the presence orabsence of the iron chelator DFO (10 μM) to mimic the effects ofhypoxia. Jurkat tumor cells were left unstimulated or were stimulatedwith PMA (50 ng/mL) and the calcium ionophore A23187 (250 ng/mL) for 16hours in the presence or absence of CoCl₂ (100 μM) to mimic the effectsof hypoxia. The cultured PBMC or Jurkat cells' HIF-1α protein levelswere assessed in 5 μg of nuclear extracts by Western blot. (FIG. 4).

Stimulation with PMA and Ionophore increased the levels of HIF-1αexpression seen in both primary leukocytes and tumor cells. PX-478reduced the HIF-1α expression in both the cell line and freshly isolatedPBMC in a dose and time dependent manner. Freshly isolated PBMC wereincubated for 16 hours in the presence of 10 μM DFO, and stimulated withPMA (50 ng/mL) and Ionophore (A23187; 250 ng/mL) in RPMI supplementedwith 10% FCS, 50 U/mL IL-2, and 5 ng/mL IL-15. PX-478 was added at 50 or100 μM for an additional 4 hours before 5 μg of nuclear extracts wereanalyzed HIF-1α protein expression by Western Blot. Jurkat cells wereincubated with DFO (10 μM) for 16 hours before the addition of either 50or 100 μM PX-478. After 4-hour incubation, 5 μg of nuclear extracts wereanalyzed HIF-1α protein expression by Western Blot. Reduction in HIF-1αexpression levels was seen within 4 hours at high concentration (50-100μM) PX-478 (FIG. 5A).

To test detection of PX-478 inhibition of HIF-1α upregulation, the CD4+T cell tumor cell line Jurkat was pre-treated with varying doses ofPX-478 for 30 minutes prior to exposure to DFO for 4 hours to induce HIFexpression. The cells were then harvested, lysed and the nuclearextracts were analyzed by Western blot for HIF-1α protein expression(FIG. 5B). At a concentration of 50 mM, PX-478 was able completelyinhibit HIF accumulation in the Jurkat cells. According to one aspect ofthe invention, therefore, PX-478 treated patients can likewise bemonitored for the ability of an HIF inhibitor to inhibit theupregulation of an HIF, e.g., HIF-1α. In lieu of doing an invasiveprocedure to procure tumor biopsy specimens before and after PX-478treatment, PBMC can be used as a surrogate biomarker to determine if thePX-478 blood levels are high enough to inhibit HIF-1α upregulation inthe presence of hypoxia mimicking chemicals such as DFO or CoCl₂.

Example 5 Development of Flow Cytometric Based Assay for Detection ofHIF-1α in PBMC and Leukemic Cell Lines.

Intracellular Staining of Peripheral Blood Mononuclear Cells (PBMC) anda Jurkat Leukemia Cell Line. Western blot analysis of HIF-1α requireslarge numbers of cells and is time consuming. These attributes makes itimpractical as a method to screen or follow patients being treated byHIF-1α inhibitors in the clinic. Therefore, we developed a flowcytometric assay that could be used to quantitate HIF-1α upregulationand/or inhibition in leukemic cells or peripheral blood mononuclearcells. This assay provides more rapid analysis of clinical samplesrequiring far fewer cells than the standard Western blot procedure.

Cells were fixed with 1.5% formaldehyde for ten minutes, permeabilizedwith ice cold methanol for 20 minutes, and stained with 1.7 μg/mL of themouse monoclonal anti-HIF-1α antibody (BD Bioscience, San Diego, Calif.)or 1 μg/mL rabbit polyclonal anti-HIF-1β (Arnt 1) antibody (Santa CruzBiotechnology, Santa Cruz, Calif.) for 30 minutes at 4° C. The cellswere washed and stained with the secondary (3.75 μg/mL finalconcentration) FITC-conjugated F(ab′)₂ goat anti-mouse IgG antibody(Jackson ImmunoResearch, West Grove, Pa.) or a 1:3000 dilution ofAlexaFluor-conjugated anti-Rabbit IgG (gift from Garth Powis). After 25minute incubation at 4° C. in the dark, the cells were washed,resuspended in 1% formaldehyde in PBS and analyzed for protein contentwithin 24 hours on a FACScan flow cytometer (BD Biosciences, San Jose,Calif.).

As shown in FIG. 6A, HIF-1α expression of Jurkat cells under normoxia(purple filled histogram, no DFO exposure) was compared to cells grownin DFO-mediated hypoxia (open histograms) in the presence or absence ofthe HIF-α inhibitor, PX-478. After a 5 hour exposure to DFO (50 μM),PX-478 was added at 0 (green line), 12.5 (blue line), 25 (black line),or 50 μM (orange line) final concentrations for a further 16 hourincubation before the cells were harvested and analyzed for HIF-1αexpression by flow cytometry. Background staining is shown fornon-specific mouse IgG control antibody (red line). FIG. 6B gives agraphic representation of the percent of cells with HIF-1α staining overIsotype control for each condition. The results show that after 16 hourincubation with 25 μM of the HIF-1α inhibitor PX-478, a 50% reductionwas seen in the HIF-1α protein expression in Jurkat tumor cells.

The flow cytometric assay was able to detect the HIF-1α upregulation ofHIF-1α due to true hypoxia (1% O₂), DFO mediated hypoxia (50 μM DFO) orto stimulation alone in PBMC isolated from a normal donor compared tocells incubated in normoxia (20% O₂). The subsequent block of HIF-1αupregulation after a 16 hour exposure to 25 or 50 μM PX-478 under allthree conditions is shown in FIG. 7A. We were also able to show that theconstitutively expressed HIF-1β protein levels were not significantlyimpacted by the HIF-1α inhibitor PX478 by staining for HIF-1β protein asdescribed in the methods above. (FIG. 7B). The present invention isuseful in determining the expression between HIF-1α expression and otherproteins of interest that may or may not be the intended target of thedrug being tested.

a particularly useful aspect of the present invention is the ability tomonitor the effective dose of drug needed to inhibit HIF expression oractivity in individual patients. To demonstrate that the assay can beused to monitor a difference in HIF-1α expression before and aftertreatment in a dose dependent manner, we spiked whole blood from anormal donor with increasing concentrations of PX-478. After a two hourincubation, the PBMC were isolated as described above by densitygradient centrifugation and after extensive washing were incubated inthe presence of stimulatory PMA and Ionophore for 16 hours. Theresulting cells were then fixed, permeabilized and stained as describedin the methods section. The flow cytometric analysis is depicted in FIG.8A. After only a two hour exposure to PX-478, there was a significantinhibition of HIF-1α upregulation by the lymphocytes. A time courseshowed that even a 30 minute exposure of whole blood to 100 μM PX478gave maximal blocking of HIF-1α upregulation (FIG. 8B).

The current study explored the use of HIF-1α upregulation in peripheralmononuclear blood cells as a means to follow HIF-1α inhibition by PX-478in a clinical setting. It is known that HIF-1α plays a critical role inthe survival and function of activated lymphocytes and has been found tobe strongly expressed in highly proliferative acute lymphocyticleukemias. Upregulation of HIF-1α protein expression in freshly isolatedperipheral blood mononuclear cells (PBMC) and the Jurkat acute leukemiatumor cell line, in vitro was accomplished using deferoxamine (DFO), aniron chelator, or CoCl₂ in combination withphorbol-12-myristate-13-acetate (1 or 50 ng/ml) and ionophore A23187(250 ng/ml) for 16-24 hours under normoxia. This treatment produced asimilar upregulation of HIF-1α as 1% oxygen exposure. It was found thatthe HIF-1α inhibitor, PX-478 reduced the HIF-1α expression in both thecultured cell line and freshly isolated PBMC to background levels within4 hours in a dose response fashion. HIF-1β is not affected by PX-478.Flow cytometry can be used to measure HIF-1α induction and inhibition byHIF-1α inhibitors, as well as measure levels of other proteins such asHIF-1β. PBMCs from freshly isolated whole blood exposed to PX-478 showedHIF-1α inhibition, as measured by flow cytometry. Flow cytometry tomonitor HIF-1α in PBMCs during PX-478 drug treatment of cancer patients,therefore, can provide a surrogate biomarker assay for clinicalefficacy.

Example 6

Identifying Subjects that will Respond to HIF-1α Inhibitors.

PBMC Isolation and Stimulation. Peripheral Blood Mononuclear Cells(PBMC) were isolated from heparinized blood of healthy volunteers oradvanced cancer patients using Histopaque-1077 (Sigma-Aldrich) densitygradient centrifugation. The PBMC were washed in PBS three times at lowspeed (200×g) to remove contaminating platelets. The resulting PBMC wereincubated overnight at 2-4×10⁶ cells/mL in 1 mL AIM-V medium(Invitrogen, Carlsbad, Calif.) supplemented with 5% FCS, 5 ng/mL IL-15and 100 U/mL IL-2 (Peprotech, Rocky Hill, N.J.) in 48 well tissueculture plates. Leukocyte activation was induced with 1 ng/mL phorbolmyristate acetate (PMA) (EMD Biosciences, La Jolla, Calif.) and 250ng/mL calcium ionophore A23187 (Sigma) and 50 μM deferoxamine (DFO) wasused to simulate hypoxia. After 16 hour incubation at 37° C. in 5.5%CO₂, the cells were harvested for HIF-1α protein analysis by flowcytometry or Western Blot.

Intracellular and Extracellular Staining of Peripheral Blood MononuclearCells (PBMC). Cells were fixed by adding 81 μL of 37% formaldehyde to 2mL of harvested cells for a final concentration of 1.5% formaldehyde andincubated for ten minutes at room temperature. The cells were spun downand the pelleted cells were permeabilized by adding 500 μL ice cold 100%methanol dropwise while vortexing, then incubating on ice for 20minutes. The cells were washed twice with 1 mL FACS Buffer (PBS+1% BSA)and stained with 1.7 μg/mL of the mouse monoclonal anti-HIF-1α antibody(BD Bioscience, San Diego, Calif.) in 100 μL FACS Buffer for 30 minutesat 25° C. The cells were washed twice with 2 mL FACS Buffer beforestaining with the secondary (3.75 μg/mL final concentration)FITC-conjugated F(ab′)2 goat anti-mouse IgG antibody (JacksonImmunoResearch, West Grove, Pa.) for 25 minutes at 4° C. in the dark.For samples requiring extracellular staining the cells were washedextensively (three times with 2 mL FACS Buffer) after HIF-1α staining,then incubated for 20 minutes at 4° C. with 5 μl of anti-CD3-PE-Cy5 (BDBiosciences) and anti-CD19-PE antibody (BioLegend, San Diego, Calif.) ina 100 μl FACS Buffer. The cells were then washed and resuspended in 200μL of 1% formaldehyde in FACS Buffer for analysis within 24 hours on aFACScan flow cytometer (BD Biosciences, San Jose, Calif.).

As shown in FIG. 9, HIF-1α accumulation in PBMCs was seen when the cellswere stimulated and could typically be enhanced under hypoxia(responder). However, it was found that donors whose PBMCs did notupregulate HIF-1α upon stimulation alone required a second signal suchas hypoxia to visualize this protein's accumulation by flow cytometry(FIG. 9; non-responder). The absence of HIF-1α accumulation withPMA/ionophore stimulation alone in the non-responder was confirmed byWestern Blot (FIG. 10).

PX-478 added directly to whole blood of two non-responders for 1 hourbefore the isolation of PBMCs and subsequent stimulation withPMA/ionophore and DFO had no effect on the HIF-1α levels under the sameconditions that lowered the HIF-1α expression of two other normal donorsby at least about 50% (FIG. 11). Whole blood was isolated from normaldonors that upregulate HIF-1α upon stimulation with PMA/Ionophore alone(responders) or that require hypoxia in addition to PMA/Ionophorestimulation (non-responders) in order to upregulate HIF-1α expression.The whole blood was spiked with PX-478 (100 μM) for 1 hour at 37° C.before PBMCs were isolated by density gradient centrifugation, washedextensively, and incubated with PMA/ionophore and DFO (50 μM) (responderA, and non-responders A & B) or with PMA/ionophore alone (responder B)overnight. The resulting PBMCs were harvested, fixed, permeabilized andstained for HIF-1α expression either in the live lymphocyte gate(responder A & B and non-responder A), or in the CD3+ T cell subset(non-responder B). The cells were analyzed by flow cytometry for thepercent gated cells expressing HIF-1α.

Example 7 Feasibility of Using Flow Cytometric Assay to Detect HIF-1αRegulation in Advanced Cancer Patients.

Developing anti-cancer drugs requires working with a unique patientpopulation that typically has undergone multiple courses of chemotherapyand has decreased white cell counts. Therefore, the feasibility of usingthe flow cytometric HIF-1α assay as a surrogate marker in a clinicaltrial setting was tested in three separate advanced cancer patients.Peripheral blood (18 mL) from two patients with advancedgastrointestinal cancer was used to isolate PBMCs. As described above,PBMCs from 2 ten mL heparin coated tubes were isolated by the standarddensity gradient method, cryopreserved, and stored in liquid nitrogen.The cells were thawed on the day of the assay, washed, then stimulatedwith PMA (1 ng/mL) and ionophore (250 ng/mL) with or without DFO (50 μM)for 16 hours (at 37° C., 5.5% CO₂) in AIMV media containing 5% FCS, 100U/mL IL-2 and 5 ng/mL IL-15. The cells were then harvested, fixed,permeabilized, and stained for HIF-1α expression in the total gatedlymphocyte population (FIG. 12A). Cells in the live lymphocyte gate wereanalyzed on the FACScan flow cytometer for percent cells expressing theHIF-1α protein. Peripheral blood mononuclear cells from a third cancerpatient was incubated for 16 hours with or without stimulation (1 ng/mLPMA and 250 ng/mL Ionophore) and DFO (50 μM) at 37° C. in 5.5% CO2 (FIG.12B). The cells were then harvested, fixed, permeabilized and stainedfor HIF-1α CD3 and CD19. The cells then underwent 3-color analysis onthe FACScan flow cytometer to determine the percent of gated CD3+ Tcells and CD19+ B cells that express HIF-1α. To analyze HIF-1αexpression in either the T cell or B cell subpopulations, the HIF-1αstained PBMC were also labeled with anti-CD3 (T cells) and anti-CD19 (Bcells). Each subpopulation was analyzed separately for HIF-1α expressionusing 3-color analysis on the FACScan flow cytometer (FIG. 12B).

Our results indicate that it is possible to detect HIF-1α upregulationin an advanced cancer patient's PBMCs. In order to enhance HIF-1αdetection and to better determine the effects the drug has on theprotein's upregulation, gating specifically on the CD3 positive T cellsprovides cleaner and more sensitive data.

Analyzing HIF-1α suppression can be a useful biomarker for any drug thatdirectly or indirectly target HIF-1α protein expression. PBMCs isolatedfrom two normal donors were stimulated with PMA (1 ng/mL) and ionophore(250 ng/mL) in the presence of DFO (50 μM) as described earlier andincubated for 16 hours with or without 25 or 50 μM PX-478 that targetsHIF-1α expression directly (FIG. 13A) or 0.5, 1, 5 or 10 μM PX-12 (FIG.13B), a drug that targets an upstream protein thioredoxin (Trx-1) knownto induce HIF-1α production as well as activity. The cells were thenharvested, fixed, permeabilized, and stained for HIF-1α expressionbefore analyzing the gated lymphocyte population on the flow cytometer.The flow cytometric assay described here was able to detect theinhibition of HIF-1α upregulation mediated by both drugs in a dosedependent fashion in both donors.

Example 8 Diagnostic Kit Composition.

Kit composition and Protocol. In order to simplify and facilitate thediagnostic techniques for monitoring or predicting therapeutic responseto HIF-modulating drugs, the use of a diagnostic kit is provided. Oneembodiment of such a kit is described below.

The kit contains a media additive comprising a concentrated stock of acytokine mixture to enhance cell survival upon stimulation (IL-2 andIL-15) called Component A. The kit further contains a concentratedcocktail containing a leukocyte stimulator and a hypoxia inducing agent(for example a stock consisting of T cell stimulators such as phorbolmyristate acetate (PMA) and Ionophore A23187 or their equivalents; PHAor its equivalent, anti-CD3 and anti-CD28 antibodies, as well as hypoxiainducing agents such as deferoxamine (DFO) or CoCl₂ or theirequivalents) called Component B. Component C comprises a mixture ofmonoclonal antibodies directed towards HIF-1α conjugated indirectly ordirectly with one type of fluorochrome (type A) and a T-lymphocytemarker (such as CD3, CD2, CD4 or CD8) labeled with a fluorochrome typeB.

Between four to ten million peripheral blood mononuclear cells orenriched or purified T cells are required for each time point to betested (i.e. pre and post treatment samples). Cells are freshly isolatedor thawed from cryogenically preserved samples. Primary lymphocyteculture media such as AIM-V (Invitrogen, Carlsbad, Calif.) supplementedwith 5% FCS is recommended for the culture of the leukocyte populations.PBMCs or enriched T cells are added at two to five million cells in 500μL media containing 1× Component A per well of a 48 well tissue cultureplate (not included in the kit). One of the two wells is designated theControl sample while the other well is designated the Activated sample.500 μl of media containing 1× Component A is added to the Control welland 500 μl of media containing 1× Component A and 1× Component B isadded to the Activated sample well. The cells are then incubatedovernight (8-20 hours). The cells are harvested, fixed and permeabilizedas described in the invention or by some comparable method.

In some embodiments, the responsiveness of the cells to an HIF-1αinhibitor is tested. Direct or indirect inhibitors of HIF-1α (such asPX-478 or PX-12) can be added at physiologically active concentrationsto activated sample wells. These samples can be analyzed as describedabove for HIF-1α expression and the extent of target inhibition in vitrocan be determined. In the case of monitoring in vivo patient response totherapies aimed at inhibiting HIF-1α, patient's peripheral bloodleukocytes or isolated lymphocytes drawn before and after treatment canbe analyzed as described above and compared to determine the extent towhich the treatment is inhibiting HIF-1α. To test which patients may beresponsive to a drug suppressing HIF-1α such as PX-478, whole blooddrawn from patients can be spiked with physiological levels of the drugand incubated at the optimal time and temperature before isolating theperipheral blood through gradient density centrifugation or somecomparable method. These cells can then be used to follow the abovemethod to determine if the drug being tested is inhibiting HIF-1αupregulation normally induced under these conditions.

The fixed and permeabilized cells are stained as described in theinvention or by some other comparable method. Such double staining isused in order to identify T lymphocytes that have increased levels ofHIF-1α expression. The numbers of double stained lymphocytes are thendetermined by means of flow cytometry. A decreased percentage of Tlymphocytes expressing HIF-1α after drug treatment compared to thebefore treatment sample will according to the invention identifyresponders to therapy.

While preferred embodiments have been described in detail, variationsmay be made to these embodiments without departing from the spirit orscope of the attached claims.

SEQUENCE LISTING

NOT APPLICABLE

1. A method of determining efficacy of a HIF-1α inhibitor therapycomprising: obtaining a first blood sample from said individual;exposing said first blood sample to a first leukocyte stimulating agent;measuring a level of HIF in said first blood sample; identifying saidindividual susceptible to treatment, wherein elevated levels of HIFindicate said individual's susceptibility to said treatment with saidHIF-1α inhibitor; administering a HIF-1α inhibitor to said individual;obtaining a second blood sample from said individual; exposing saidsecond blood sample to a second leukocyte stimulating agent; measuring asecond level of HIF expression in said second blood sample, whereindecreased levels of HIF indicates effective HIF-1α inhibitor therapy. 2.The method of claim 1, wherein said leukocyte stimulating agent is a Tcell stimulating agent.
 3. The method of claim 1, wherein said leukocytestimulating agent is selected from phorbol myristate acetate, calciumionophore, concanavalin A, anti-CD3 antibody, anti-CD28 antibody, PHA,SEA, SEB, and combinations thereof.
 4. The method of claim 1, whereinsaid HIF-1α inhibitor is selected from PX-478, PX-12, 2-ME2, topotecan,camptothecin and combinations thereof.
 5. A method of determiningefficacy of a HIF-1α inhibitor therapy in an individual comprising:obtaining a first blood sample from said individual; measuring a firstlevel of HIF in said blood sample; administering a HIF-1α inhibitor tosaid individual; obtaining a second blood sample from said individual;exposing said blood sample to a leukocyte stimulating agent and hypoxicconditions; and measuring a second level of HIF in said second bloodsample, wherein decreased levels of HIF indicates effective HIF-1αinhibitor therapy.
 6. The method of claim 5, wherein said leukocytestimulating agent is a T cell stimulating agent.
 7. The method of claim5, wherein said leukocyte stimulating agent is selected from phorbolmyristate acetate, calcium ionophore, concanavalin A, anti-CD3 antibody,anti-CD28 antibody, PHA, SEA, SEB, and combinations thereof.
 8. Themethod of claim 5, wherein said HIF-1α inhibitor is selected fromPX-478, PX-12, 2-ME2, topotecan, camptothecin and combinations thereof.